The tissue engineering field is advanced and a variety of tissue equivalents have been developed. Not only do these equivalents vary in the material that is used as a scaffold, they also vary in the type and source of cells which are seeded on the scaffold. Skin equivalents are one area of tissue engineering that has witnessed significant research and commercial development.
Modern skin equivalents may be made using a variety of materials and methods. For example, a cultured skin may be prepared by culturing human fibroblasts in collagen gel, followed by inoculating and culturing human keratinocytes on the gel after the gel has shrunk (U.S. Pat. No. 4,485,096). Another skin equivalent is prepared by inoculating and culturing human fibroblasts on nylon mesh, followed by inoculating and culturing human keratinocytes thereon when the pores of the mesh are filled up with secreted materials from the fibroblasts (Slivka, S. R., L. Landeen, Zimber, M., G. K. Naughton and R. L. Bartel, J. Invest. Dermatol., 96: 544A, 1991). Alternatively, a skin may be prepared by inoculating and culturing human fibroblasts on a collagen sponge, followed by a laminating collagen gel or film that is inoculated with human keratinocytes thereon (J. Jpn. P. R. S., 10, 165-180 (1990) and Japanese Examined Patent Publication No. 47043/1995).
The advanced nature of skin equivalents is demonstrated by the fact that a number of skin equivalents are commercially available. Commercial skin equivalents include EpiCel™ (which lacks a dermal component and uses the patient's own cultured keratinocytes) Integra™ (which uses a collagen-glycosaminoglycan (GAG) matrix to provide an acellular dermal component and uses a thin epidermal autograft), AlloDerm™, (which uses a dermal matrix and a thin epidermal autograft), DermaGraft™ (which uses a polyglycolic acid/polylactic acid (PGA/PLA) matrix and allogeneic human fibroblasts for the dermis), Hyaff/LaserSkin™ (which uses hyaluran and fibroblasts for the dermis, and hyaluran and the patient's own keratinocytes for the epidermis), and PolyActive™ (which uses polyethylene oxide/polybutylthalate (PEO/PBT) and the patient's own fibroblasts for the dermis, and the patient's cultured keratinocytes for the epidermis).
Other commercially available skin equivalents include ApliGraft™, which uses collagen gel and allogeneic fibroblasts for the dermis, and cultured allogeneic keratinocytes for the epidermis, Comp Cult Skin™ or OrCel™, which uses collagen and allogeneic fibroblasts for the dermis, and cultured allogeneic keratinocytes for the epidermis, and TransCyte™, which uses allogeneic fibroblasts for the dermis and a synthetic material, BioBrane™, for the epidermis.
Skin equivalents have been used to treat a variety of skin defects. Successful regeneration of skin has been observed for injuries such as burns and skin disorders that result from a disease such as diabetic ulcers. The use of cellular scaffolds (i.e. skin equivalents) is not limited to therapeutic tissue regeneration as they are also applied to cell-based assays and tissue culture systems.
The production of therapeutic tissue models using fibroblasts in combination with other cells, such as epidermal keratinocytes presents certain challenges. For example, although fibroblasts provide growth factors and other cell-to-cell contacts that facilitate cell division, their proliferation may outpace epidermal cell division resulting in a culture that is overgrown with fibroblasts. This is clearly undesirable as therapies aimed at the regeneration of epidermal tissues must be carried out using carriers rich in epidermal cells. One means of preventing the overgrowth of fibroblasts involves plating the epidermal cells with irradiated 3T3 (mouse) fibroblast cells (Rheinwald and Green, Cell, 6, 331-334, November 1975). However this technique requires the presence of dermal components which is undesirable in therapeutic applications. Another approach for producing skin equivalents with a high number of keratinocyte precursors is to seed the cell scaffold with more keratinocytes than fibroblasts. However, when precursor keratinocytes are cultured without fibroblasts, they produce greater levels of collagenase making the seeding of collagen scaffols with keratinocytes alone impossible.
What is needed in the art therefore is a tissue equivalent having therapeutically appropriate proportions of keratinocyte and fibroblast cells. Also needed in the art is a means for producing a collagenase-inhibited population of keratinocyte stem cells which are capable of being expanded on a collagen scaffold in the absence of fibroblasts.